PB1786: TRINARY FUSION AND LIGAND BINDING DOMAIN TRUNCATION AS RECURRENT AND CRUCIAL MOLECULAR CHARACTERISTICS IN RARG-DRIVEN ACUTE PROMYELOCYTIC LEUKEMIA AND RENDER UNRESPONSIVE TO ALL-TRANS RETINOIC ACID
Authors: Xiaosu Zhou, Xue Chen, Jiaqi Chen, Panxiang Cao, Ping Wu, Jiancheng Fang, Xiaoli MA, Tong Wang, Mingyue Liu, Fang Wang, Yang Zhang, Lili Yuan, David Jin, Ming Liu, Wen Teng, Ruijie Tang, Xian Zhang, Hongxing Liu
Journal: HemaSphere (2023)

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: All-trans retinoic acid (ATRA) and arsenic have been used with great success in the treatment of PML::RARA fusion gene (FG) positive acute promyelocytic leukemia (APL). There are still PML::RARA negative cases manifest like APL, that is, variant APL (APLv). These cases often carry rare FGs involved in RARA, RARB, or RARG. So far, 24 RARG-FG positive APLv (RARG-APL) cases have been reported, increasing year by year, thanks to the application of transcriptome sequencing (RNA-seq) and whole genome sequencing (WGS). Almost all reported cases tried ATRA treatment, but all evaluable cases were resistant. On the contrary, cells transfected with artificial X::RARG fusion protein responded well to ATRA in vitro. Aims: To decipher the undiscovered traits of RARG-FGs and the critical molecular abnormalities in RARG-APL. Methods: The characteristics of RARG-FGs were analyzed by WGS, RNA-seq, and RT-PCR. Sequence-specific primers were designed to determine whether two fusion sites were located on the same cistron. The biological effects and responsiveness to ATRA of fusion proteins were evaluated in vitro. Results: In 2022, we admitted one APLv case (#1) who was resistant to ATRA and chemotherapy. In this case, we identified a trinary fusion of PRPF19-e1_4::RARG-e4_9::PRPF19-e5_16 (e, abb. of exon). The RARG 3’ and 5’ fusions were confirmed to be in the same cistron. That is, the fusion is the insertion of RARG-e4_9 between PRPF19-e4 and e5, rather than forming two transcripts of PRPF19-e1_4::RARG-e4_10 and RARG-e1_9::PRPF19-e5_16. This case reminds us of a case (#2) with NPM1::RARG::NPM1 fusion that we reported [Br J Cancer.2019;120:1023-1025], the only case reported as RARG trinary fusion till now. We then performed analysis on archived samples of one more APLv case (#3) and identified HNRNPC:: RARG::HNRNPC fusion. We also reanalyzed one case (#4) reported by other groups for which RNA-seq data was available [Hematology.2022;27:518-522]. This case was reported with the RARG-e9::HNRNPM-e3 fusion, but our ancillary analysis further confirmed the HNRNPM-e12::RARG-e1 splicing. The untranslated region (UTR) of RARG-e1_3 confers a derived coding sequence, and the reading frame of RARG-e4_9 was retained in this trinary fusion. The involvement of UTR caused bioinformatic analysis failure to report in-frame fusion, resulting in missed detection. RARG-e10 deletion was a strictly recurrent event in all 4 cases. RARG-e10 encodes helix 11_12 (H11_12) of its ligand binding domain (LBD), which plays a pivotal role in response to ATRA [Structure.2019;27:1270-1285.e6]. In principle, the RARG trinary fusion protein with LBD truncation will lose responsiveness to ATRA. Our in vitro experiments also confirmed that cells transfected with chimeric protein with intact RARG LBD responded well to ATRA, while chimeric proteins with RARG LBD-H11_12 truncation were unresponsive to ATRA even at high concentrations. Summary/Conclusion: We revealed that trinary fusion and LBD truncation as recurrent and crucial molecular features in RARG-APL and render unresponsiveness to ATRA. RARG 5’ translocation leads to aberrant expression, with variation in the splicing site (intron 3 or further upstream). RARG 3’ splicing is highly recurrent at exon 9, resulting in LBD truncation, which plays a pivotal role in leukemogenesis and renders unresponsiveness to ATRA. This trait was ignored previously due to the complexity of trinary fusion, and the inertial only focus on RARG 5’ splicing. We are now recruiting multicenter RNA-seq data to further validate this finding.Keywords: Gene fusion, AML, Retinoic acid receptor (RAR), APL